The invention concerns scaffold matrices for supporting three-dimensional tissues and systems for maintaining three-dimensional viable tissues.
The following publications are believed to be relevant as background of the invention.
1. WO 98/22573
2. U.S. Pat. No. 4,880,429
3. U.S. Pat. No. 4,108,438
4. U.S. Pat. No. 5,843,182
5. Mikos, A. G., Sarakinos, G., Leite, S. M., Vacanti, J. P., and Langer, R., xe2x80x9cLaminated three-dimensional biodegradable foams for use in tissue ingineeringxe2x80x9d, Biomaterials, 14:323-330, 1993.
6. Hutmacher, D., Kirsch, A., Ackeman, K. L., and Huerzeler, M. B., xe2x80x9cMatrix and carrier materials for bone growth factorsxe2x80x94state of the art and future prospectives in: Stark, G. B., Horch, T, Tancos, E. (eds). Biological Matrices and Tissue Reconstruction. Springer Verlag, 1998, pp. 197-203.
7. Kandel, R. A., Chen, H., Clark, J. and Renlund, R., Transplantation of cartilaginous tissue generated in vitro into articular joint defects. Biotechnol, 23:565-577, 1995.
Cartilage is a specialized form of connective tissue composed of cells and matrix. The cartilage cells synthesize matrix and become encased in cavities (lacunae) within it. The matrix is composed of fibers embedded in ground substance and endows cartilage with its specialized physico-chemical properties.
Trauma, single or repetitive, and minute imbalance in joint stability are the most known causes of damage and degeneration of articular cartilage, that leads to pain, chronic disability and ultimately to joint failure. The current options for treatment provide temporary improvement of symptoms and function, however, there is no full restoration of joint performance. Prosthetic joint replacement is currently the ultimate and the most commonly employed treatment. Modem biological grafting is the other alternative for resurfacing the damaged joint, but is still imperfect.
A large number of candidate grafts have been studied for enhancing the repair of cartilage defects which include: (i) Osteochondral graft (autografts or allografts); (ii) Intact cartilage grafts; (iii) Growth plate; (iv) Isolated allogeneic chondrocytes; (v) Cultured autologous chondrocytes (dedifferentiated) (vi) Periosteum; (vii) Perichondrium; (viii) Bone marrow mesenchymal derived cells and (ix) Synovial membrane derived cells.
Another approach was the attempt to use natural occurring or synthetic biodegradable scaffolds which support three-dimensional growth of cartilage cells. The scaffolds may be impregnated with cells, which together with the scaffold form the graft. Alternatively, the scaffold may initially be devoid of impregnated cell, and endogenous cells from the patient are expected to migrate into the scaffold after its implantation.
Examples of such scaffolds are: (a) Fibrin polymers; (b) Collagen Type I; (c) Natural hyaluronic acid (HA) and chemically modified HA and (d) Synthetic biopolymers either biodegradable e.g. polylactic acid, polyglycolic acid or non-biodegradable (e.g. alginic acid). However, none of the above scaffolds can induce regeneration of hyaline-like cartilage. Fibrin adhesive polymers tend to induce dedifferentiation and thus do not permit production of functional tissue. Collagen Type I has no inherent chemotactic ability for chondrocytes, but stimulates proliferation of fibroblasts. Thus, instead of encouraging migration of chondrocytes, the tissue formed in this scaffold tends to be fibrous. Hyaluronic acid can stimulate chondrogenic differentiation, but does not stimulate chondrocytes proliferation. Alginic acid is a foreign sea weed derived carbohydrate and thus might induce an antigenic reaction, and furthermore it is not biodegradable. Polyglycolic and polylactic acid scaffolds do not support good hyaline cartilage regeneration due to acidic conditions formed during their degradation.
Damaged or missing hyaline cartilage is frequently repaired by transplantation of homografts. Homografts are immunologically privileged since the matrix acts as a barrier that permits only limited diffusion of low-molecular weight substances and contains an anti-angiogenesis factor to prevent invasion of host blood vessels and fibroblasts.
Various culturing systems have been developed for maintaining the viability and growth of tissues in culture. Generally, these are divided into static and perfusion bioreactors. Perfusion bioreactors are reactors which essentially keep constant, growth permissible conditions (such as nutrition, gas composition, temperature, pH, etc.) in which the growth fluid medium is constantly perfused in and out of the system. Typically, perfusion is carried out by utilizing a constant velocity flow of the medium.
By a first aspect, the present invention concerns a scaffold for use as growth supportive base for cells and tissue explants from three-dimensional tissue, comprising a naturally derived connective or skeletal tissue which has been treated for elimination of cellular and cytosolic elements, and which has been modified by cross-linking with an agent selected from the group consisting of: hyaluronic acid, proteoglycans, glycosaminoglycan, chondroitin sulfates, heparan sulfates, heparins and dextran sulfates.
By a second aspect, the present invention concerns a scaffold for use as growth supportive base for cells and tissue explants from three-dimensional tissue, comprising a naturally derived connective or skeletal tissue which has been treated for elimination of cellular and cytosolic elements, and which is formed of flakes having a size below 1000xcexc, which are attached to each other, the scaffold having a porosity of at least 85%, most preferably a porosity of 95%-98%.
By a third aspect the present invention concerns a scaffold for use as growth supportive base for cells and tissue explants from three-dimensional tissue, comprising an aggregate of at least 3, preferably 7-8 embryonal epiphyses, obtained from a third to midterm old fetuses (11 days old chick embryo, or 17-22 weeks human embryos). The aggregates are formed spontaneously, when several individual epiphyses are attached to each other, due to the presence of mesenchymal progenitor cells in the periphery of the epiphysis, cells which feature high expression of adhesive molecules such as integrins, cadherins and CAMs.
It has been found that such scaffolds according to the first, second and third aspects of the invention, have the properties of encouraging cells"" adherence both to the matrix and to other cells as well, and enablement of propagation of cells. It was further found that the scaffolds of the invention supports chondrocyte proliferation at the expense of fibroblasts, resulting in a hyaline-like reparative repair tissue. Cross-linking with the agents specified above in connection with the first aspect gives the scaffold an additional mechanical strength and produces a substance which is less brittle with prolonged biodegradation time.
The term xe2x80x9cscaffoldxe2x80x9d in the context of the first and second aspects of the present invention refers to the connective/skeletal tissue which has been treated for elimination of cellular and cytosolic elements. In accordance with the first aspect of the invention, the scaffold has been modified by cross-linking as described above. In accordance with the second aspect, the scaffold is composed of attached flakes having a size of less than 1000xcexc and having porosity of at least 85%. This term also refers in accordance with the first and second aspect to such a construct containing additional agents such as adhesive molecules or growth factors.
The term xe2x80x9cflakesxe2x80x9d refers to particles or chips produced as a result of crushing the connective tissue, to particles of a size less than 1000xcexc.
In accordance with the third aspect of the invention the term xe2x80x9cscaffoldxe2x80x9d concerns freshly aborted human epiphyses from around midterm (18xc2x14 weeks), which are composed of mesenchymal progenitor stem cells and committed chondrocytes. At least 3 and preferably 7-8 epiphyses have been fused to form aggregates which could be formed spontaneously due to the adhesive properties of the mesenchymal cells. The aggregates were formed by growth in either static culture or in the system of the invention as will be described hereinbelow.
The term xe2x80x9cthree-dimensional tissuexe2x80x9d (3D tissue) refers to any type of tissue which has an orderly three-dimensional structure, i.e., is not naturally present in the body in the form limited to a single layer of cells or lamina, but has a structure which is spatially ordered. Examples of three-dimensional tissue are: mesenchymal tissue, cartilage and bone tissue, liver tissue, kidney tissue, neuronal tissue, fibrous tissue, dermis tissue etc. Another three-dimensional tissue is the whole embryonal epiphyseal organ derived from embryos at a post limb-bud stage.
The naturally derived connective or skeletal tissue is, in general, a tissue that was derived from mesenchymal tissues that expresses, temporarily or continuously. fibroblast growth factor receptor 3 (FGFR3). Examples of such tissue are mainly members of the chondrogenic and the osteogenic anlagen, as well as the residual mesenchymal stem cell reservoirs found in tissues all along life, ready to carry wound healing, repair and regeneration tasks. Another example of connective or skeletal tissue is epiphyseal tissue, periosteal and perichondrial flaps that contain massive growth factors, and bone marrow.
In order to turn a tissue into a scaffold in accordance with the first and second aspects of the invention, the tissue should be treated for elimination of cellular and cytosolic elements such as: DNA, RNA, proteins, lipids, proteoglycans and in general most elements of the cells which are immunogenic, as well as treated for removal of calcification-mineralization centers. Methods for elimination of the above cellular and cytosolic elements are in general known in the art, preferably the elimination is achieved by alternating freezing and thawing cycles in distilled water serving as thoroughly washes, which eliminate lysed cells"" contents, followed by incubationxe2x80x94evaporation in alcohol at 45-55xc2x0 C. within an air blowing incubator.
The naturally derived connective or skeletal tissue treated as described above for elimination of cellular and cytosolic components, in connection with the first aspect of the invention, is preferably further treated for producing higher porosity, of the intact tissue, is by the production of pores in a controlled manner. The treatment may be mechanical, for example, by hammering the tissue on a scraper device, or by hammering a metal brush into the tissue (e.g. epiphyseal tissue).
Alternatively, the treatment for producing porosity may be a chemical extraction process carried out by exposing the tissue, for a controlled amount of time in a controlled environment, to chemical agents capable of partial degradation of the tissue. In addition or alternatively, the treatment for producing porosity may be carried out by exposing the tissue to enzymatic agents such as proteolytic enzymes, capable of partial degradation of the tissue. Example of such chemical agents which can produce pores in the tissue are guanidinium chloride. The pores should have preferably a size of 10-500xcexc, most preferably 20-100xcexc.
The agents either specified in above (i.e. hyaluronic acid, proteoglycans, glycosaminoglycan, chondroitin sulfates, heparan sulfates, heparin and dextran sulfates) or additional agents such as adhesive molecules or growth factor moieties may be linked to the residual scaffold either by sugar cross-linking, (for example using 1% of either ribose or xylose), by carbodiimide or by 1,1 carbonyl di-imidazole. Cross-linking with the above agents is generally carried out as known in the art of coupling in organic chemistry.
In accordance with the second aspect of the invention, the porosity is an inherent property of the scaffold of the invention, as the scaffold is made of flakes which have a size of less than about 1000xcexc, attached to each other, so that the overall porosity of the scaffold is above 85%, preferably above 90%, most preferably above 98%.
Such high porosity is obtained, after elimination of cytosolic elements, as described above, by crushing the connective skeletal tissue which has been treated for elimination of cellular and cytosolic elements, to small particles, preferably having a size below 1000xcexc and then loosely attaching these small flakes to each other. One manner for such attachment, is by suspending the small flakes in alcohol, and then evaporating the alcohol in a vessel with a very large surface, resulting in a residue of material having a xe2x80x9ccrust-likexe2x80x9d characteristic, with a very high porosity.
Preferably, the scaffold of the invention is composed of several, essentially single-flake layers of xe2x80x9ccrust-like materialxe2x80x9d, arranged one on top of the other, most preferably a construct of at least 5 layers. Preferably, the layers are fused to each other at their edges, by heat-thermo treatings, for example by application of laser irradiation.
The fusions of the xe2x80x9ccrust-likexe2x80x9d material may be improved by the addition of small amounts, of 10-40% albumin solution, these layers are added in the periphery of each layer.
By another aspect, the present invention concerns a method for the production of a scaffold for use as a growth supporting base for cells and tissue explants obtained from three-dimensional tissue, the method comprising:
(i) providing naturally derived connective or skeletal tissue;
(ii) eliminating cellular and cytosolic elements from said connective tissue;
(iii) crushing the connective tissue obtained in (ii) to produce flakes having a size of less than 1000xcexc;
(iv) suspending the flakes in alcohol;
(v) evaporating the alcohol in a vessel having a large surface face, thereby producing a scaffold which has essentially no cellular and cytosolic elements, and has a porosity of at least 85%, preferably 90%, most preferably 98%.
In accordance with the present invention, it is preferable that the scaffold also contains adhesive molecules in order to enhance cell adherence to the scaffold. Example of suitable adhesive molecules are the integrins and additional extra cellular constituents known to interact, agents such as, laminin, fibronectin, hyaluronic acid, polylysine, lysozyme and collagen. The formation of collagen, for example, may be enhanced by additions of ascorbic acid and its stable derivative such as ascorbic-2-phosphate. Said adhesive can be used in accordance with all three aspects of the invention.
In accordance with the present invention, it is also preferable that the scaffold would contain endogenously or exogenously added growth factors, in order to enhance the rate of growth of the cells filling the three-dimensional space of the scaffold. Examples of suitable exogenously added growth factors are: fibroblast growth factors (FGF""s), TGF""s, BMP""s, IGF""s. The growth factor chosen should depend on the type of tissue used. It should be noted that scaffolds of natural tissues, devoid of cells and cytosolic elements may still contain endogenous growth factors, bound to extracellular matrix elements so that at times the endogenous growth factors present in the matrix are advantageous ingredients.
By one option, it is possible to formulate a prosthesis from the scaffold alone in accordance with the first and second aspects of the invention, i.e. of a scaffold devoid of cells. In such a case, the scaffold is formulated to a desired shape and is inserted into the desired location in the body of the individual, for example, a location wherein it is desired to achieve invasion of endogenous mesenchymal cells such as in the knee joint.
The prosthesis is maleable and can be shaped as either a flat sheet of several millimeters in thickness or any other three-dimensional shape adapted to the shape of the lesion. As explained above in connection with the second aspect of the invention, the xe2x80x9ccrust-likexe2x80x9d scaffold having a porosity of at least 85%, can be formed by stacking several single-flake layers on top of the other, preferably using at least 5 such layers. Most preferably the edges of the layers may be fused to one another to provide mechanical support, for example, by fusing them using adhesives with or without laser irradiation creating heat (60-70xc2x0 C.).
Alternatively, the prosthesis, can, a priori, prior to implantation contain embedded (impregnated) cells, for example cells grown originally as monolayers or multi-layers on filters (Millicell cell culture [PICMORG50] inserts for use in organotypic cultures, 30 mm, low height, Millipore Corp. Bedford, Mass, USA), and placed 5-10 units in the device described further below, or tissue explants to allow their fast anchorage and integration into bone and cartilages.
The cells impregnating the prosthesis should preferably be from an autogeneic source, but can also be of an allogeneic source, as cartilage has a sort of an immunoprivilage.
The scaffold of the invention according to both the first and second aspects of the invention (after treatment for elimination of the endogenous cellular elements) can be impregnated with exogenous cells, not only for direct implantation in the body but also for prolonged in vitro growth and differentiation of various three-dimensional tissues kinds such as skin, neuronal, bony, cartilaginous, liver, pancreatic beta cell and almost of any organ or tissue in a bioreactor, while adjusting the proper medium, cocktail of growth factors and adhesive molecules.
By another aspect the present invention concerns a system for maintaining viable three-dimensional tissue.
Static cultures in regular incubators can support cell growth in monolayers, multilayers or at most few microns 50-100 micron of 3D explants. For larger 3D explants only special bioreactor devices can support growth by perfusing nutrients, gases and remove wastes.
In accordance with this second aspect, it was surprisingly found that for long-term maintenance of viable three-dimensional tissue, there is need to apply rhythmic pulses of pressure (hydrostatic, mechanical or shear force) in order to obtain optimal growth. For example for growing of an articular cartilage tissue there is an advantage in maintaining the tissue under repetitious cycles of loads and unloads of pressure in a rhythmic manner, simulating the natural growth conditions in the joint. The cellular mechanoreceptors seem to play a key role in this respect of cell growth.
The variables that can be manipulated in the system of the invention include stream flow velocity, amount (in atmospheres) of hydrostatic and/or mechanic pressure, rhythmic action periods (frequency of applications of pressure) and pausal intervals (pulses), as well as change stream direction of the medium. By this second aspect, the present invention concerns a system for the maintenance of viable tissue comprising:
(ix) a chamber for holding the tissue, the chamber""s atmosphere being kept at a relatively constant gas composition, said gas composition being suitable for maintenance of viable biological tissues;
(ii) a reservoir for holding tissue culture medium, said reservoir being in flow communication with the chamber;
(iii) a pump for circulating the medium between the chamber and the reservoir in a controlled manner; and
(iv) a pressure generator for producing rhythmic pulses of pressure on the tissue present in the chamber.
The system of the invention is suitable for any type of cells or tissues, but is especially suitable for the growth of a three-dimensional tissue, according to the definition above.
Basically, the system comprises a chamber for holding the tissue, the chamber""s atmosphere being kept at relatively constant gas and temperature composition which are suitable for maintenance of viable biological tissue, for example 5% to 10% CO2 in air at physiological temperature. This is usually achieved by placing the chamber within a larger CO2 incubator, capable of maintaining such an atmosphere, and ensuring that the atmosphere of the incubator in communication (as regards temperature and gas composition) with that of the chamber.
The system farther comprises a reservoir for holding tissue culture medium which is in flow communication with the chamber. Preferably, the size of the reservoir is about 30 to 100 times larger than that of the chamber for holding the tissue and is typically the size of 400-1000 ml. The medium in the reservoir of course contains the nutrients and various agents such as growth factors, etc. required for maintaining viability and growth of the tissue.
The system comprises a pump which circulates the growth medium between the chamber and the reservoir in a controlled manner. The pump may be a constant pump or a peristaltic pump utilizing either computerized or electrical/electronical manipulated regimes, as will be explained hereinbelow. Typically, the velocity of medium flow is in the range of 300-600 ml/min.
The pressure generator may produce mechanical or hydrostatic pressure on the tissue and may be, for example, a compressor (piston) present in the chamber, which can periodically apply pressure on the tissue present in the chamber when streaming in one direction and the pressure is released when streaming in the other direction. The compressor should be under control of a control mechanism capable of controlling the timing (frequency, pausal, etc.) and the level of compression, such as a clock or a computer mechanism.
The control mechanism would trigger the compressor, to compress the chamber thus applying rhythmic pressure on the liquid present therein, and consequently applying pressure on the tissue. In the case of a compressor, the pump""s activity may be constant so that the medium circulates between the chamber and the reservoir at a constant rate in order to improve gas exchange and nutrient availability to the tissue.
By another alternative, the pump that circulates the medium between the chamber and the reservoir is itself the pressure generator capable of producing rhythmic pulses of hydrostatic pressure on the tissue. In that case the pressure generator is the pump itself and no additional elements (such as a compressor) are required to produce the rhythmic pulses of pressure. The pump which is a peristaltic may have a built-in means for triggering rhythmic pulses. Alternatively, the pump may be connected to a control mechanism which triggers the duration, delays and frequencies of the pump such as a clock or a computer mechanism.
By alternating activities of such a pump, the medium can circulate in pulses between the medium reservoir and the chamber, thus creating rhythmic pressure pulses on the tissue. Preferably the direction of the medium flow should be changed (for example clock-wise and then counter-clock-wise) as changing the flow direction simulates best the joint""s conditions of loading and unloading. Typically change of direction should be every 1 to 3 min.
The rhythmic pulses should have a frequency of 5-300 per min., preferably 10-200 per min., most preferably 60 to 120 per min.
The hydrostatic pressure should be between 0.5 and 30 atm., preferably 1 to 10 atm, most preferably 2 to 3 atm.
The present invention concerns a method for maintaining viable tissues, cells or explants from three-dimensional tissue, comprising placing these tissues in the chamber of the above system with any of the above parameters of pressure, frequency and change of flow direction. Examples of tissue are as defined above in connection with the scaffold.
The present invention further provides a method for maintaining viable cells or tissue explants from three-dimensional tissue comprising growing a prosthesis composed of the scaffold of the invention (in accordance with both aspects) impregnated with cells in the system of the invention with conditions specified above (i.e. the parameters specified above). A preferable example is a method for growing fresh cartilaginous tissues such as embryonal epiphyseal tissue, turning to an allogeneic implant upon in vivo transplantation.